Cytoplasmic concentrations of inorganic phosphate affect the critical concentration for assembly of actin in the presence of cytochalasin D or ADP.

Skeletal muscle actin labelled with pyrene was used to measure the critical concentration (Cc) for assembly in conditions designed to approximate the ionic environment in the cytoplasm. Under these conditions (0.1 M-KCl, 2 mM-MgCl2, 1.1 mM-ATP, 0.1 mM-CaCl2, 0.5 mM-ethyleneglycol-bis(beta-aminoethylether)N,N'-tetraacetic acid, 0.25 mM-2-mercaptoethanol, 20 mM-imidazol X HCl, pH 7.0), the steady-state Cc value was estimated to be 0.07 microM (3.0 micrograms/ml), and, consistent with previous observations, the Cc increased to 0.20 microM (8.7 micrograms/ml) in the presence of 10(-6) M-cytochalasin D, and to 1.10 microM (47 micrograms/ml) after conversion of ATP to ADP using hexokinase and glucose. Addition of inorganic phosphate (Pi) at concentrations up to 20 mM caused only a slight decrease in the steady-state Cc, but at 2 mM-Pi (a reasonable estimate of cytoplasmic concentrations) the increase in Cc due to cytochalasin D was abolished, and at higher Pi concentrations there was even a slight decrease. Increasing Pi concentrations also progressively reduced the steady-state Cc for ADP-actin close to that for ATP-actin. These results are consistent with an increased affinity of ADP-actin for the polymer in the presence of Pi. To determine whether these effects of Pi were simply mass action effects on hydrolysis of bound ATP by polymerized actin, the stoichiometry of ATP hydrolysis during actin assembly was estimated and found to be at unity within the limits of experimental error and to be unaffected by Pi up to 20 mM. In addition, actin depolymerized by removal of ATP using glucose and hexokinase rapidly reassembled after addition of 20 mM-Pi. These results are interpreted by a mechanism involving the formation of ADP-Pi-actin species and are discussed in relation to the phenomenon of treadmilling and the theory of dynamic instability, and the potential for their occurrence in cells.